Antimicrobial susceptibility and molecular characterisation using whole-genome sequencing of Clostridioides difficile collected in 82 hospitals in Japan between 2014 and 2016

Abstract

We studied the antimicrobial susceptibility and the molecular characteristics using draft whole-genome sequencing of Clostridioides (Clostridium) difficile strains before and after treatment in adults with C. difficile infection (CDI) enrolled in a Phase III, randomised, nationwide study of fidaxomicin versus vancomycin in Japan (NCT02179658). C. difficile strains were cultured from stool samples collected before and after standard treatment with either fidaxomicin or vancomycin. Overall, 285 C. difficile strains were recovered, with 188 derived from CDI cases at baseline (87 patients received fidaxomicin and 101 received vancomycin). No strains isolated from episodes of CDI at baseline were shown to have reduced susceptibilities to fidaxomicin (MIC ≥1 mg/L), or resistance to vancomycin and metronidazole. Thirty-three sequence types (STs) were identified, the most common being ST17 (n=61; 32.4%), ST8 (n=26; 13.8%) and ST2 (n=21; 11.2%). Core-genome single nucleotide polymorphism analysis showed that outbreaks of C. difficile were unlikely to have occurred at each hospital. The predominant toxin gene profile was tcdA⁺tcdB⁺cdtA/cdtB^- (n=149; 79.3%). Six of 87 patients who received fidaxomicin harboured C. difficile isolates with reduced fidaxomicin susceptibilities conferred by previously described mutations, Val1143Leu/Gly/Asp in RpoB or Arg89Gly in RpoC or putative mutations, Gln1149Pro in RpoB or Arg326Cys in RpoC. Allelic exchange studies of these putative mutations were not performed. Prior to fidaxomicin use, we found no C. difficile strains with reduced fidaxomicin susceptibilities causing CDI in Japan; however, mutant strains with reduced fidaxomicin susceptibilities were detected after fidaxomicin treatment.

FIG 1

Patient flow in the phase III, randomized, nationwide study of fidaxomicin versus vancomycin in Japan. a, patients diagnosed with Clostridioides difficile infection (CDI) were positive for C. difficile toxin A, B, or both in a stool specimen obtained within 96 h before randomization. b, days 1 to 10. c, if a stool specimen was obtained from the patient within 24 h of completing the treatment (days 10 and 11), C. difficile culture was performed. d, if diarrhea occurred within 28 days (± 3 days) after treatment, C. difficile culture was performed. e, one secondary C. difficile strain was isolated unexpectedly during fidaxomicin administration. f, two secondary C. difficile strains were isolated unexpectedly during vancomycin administration.

FIG 2

Phylogenetic tree of Clostridioides difficile strains belonging to each sequence type (ST) constructed with maximum likelihood phylogenetic analysis based on single-nucleotide polymorphisms (SNPs) in the core genome, excluding estimated homologous recombination. The scale of the distances corresponds to the average number of substitutions per site and SNP. (A) Phylogenetic tree of 61 strains of C. difficile ST17. A core genome region, amounting to 58.4% (2,505,609/4,290,252 bp), was shared with the genome of a reference strain, C. difficile 630 (ST54). (B) Phylogenetic tree of 26 strains of C. difficile ST8. A core genome region, amounting to 32.0% (1,374,263/4,290,252 bp), was shared with the genome of a reference strain, C. difficile 630 (ST54). (C) Phylogenetic tree of 22 strains of C. difficile ST2. A core genome region, amounting to 34.1% (1,461,002/4,290,252 bp), was shared with the genome of a reference strain, C. difficile 630 (ST54). (D) Phylogenetic tree of 19 strains of C. difficile ST81. A core genome region, amounting to 72.6% (3,114,637/4,290,252 bp), was shared with the genome of a reference strain, C. difficile 630 (ST54). (E) Phylogenetic tree of 13 strains of C. difficile ST183. A core genome region, amounting to 41.8% (1,795,356/4,290,252 bp), was shared with the genome of a reference strain, C. difficile 630 (ST54).